Regulation of gene expression is crucial in all cells, and the importance of RNA-mediated regulation in a wide variety of cellular systems has emerged in the past decade. This project focuses on the T box riboswitch, which monitors the aminoacylation status of individual tRNA species to regulate the expression of many amino acid-related genes, including essential aminoacyl-tRNA synthetase genes, in Gram-positive bacteria. The T box riboswitch was discovered in the PI's laboratory, and most of the fundamental work describing the regulatory mechanism, and the basis for specific tRNA recognition, has been carried out in this laboratory. We have shown that tRNA recognition requires pairing of the tRNA anticodon with a single codon, the Specifier Sequence, in the leader RNA, as well as pairing of the acceptor end of the uncharged tRNA to an antiterminator element that competes with formation of the terminator helix. These pairings are necessary but not sufficient for antitermination, and we recently uncovered two new interactions, one with the tRNA elbow region and the other with the tRNA T arm; however, the residues that participate in these interactions are not universally conserved in T box RNAs, suggesting that other elements can compensate. In the next stage of the project, we will focus on how leader RNA-tRNA interactions that are crucial in some groups of T box RNAs are replaced by alternate interactions in other T box RNA-tRNA complexes, and contribute to specific cognate tRNA recognition and discrimination against non-cognate tRNAs. We have demonstrated that, in addition to regulation at the level of transcription attenuation, regulation also can occur at the level of translation initiation, and will investigate the implications of this alternate mechanism. We also will investigate systems in which multiple T box-regulated genes in the same cell interact to integrate additional physiological signals. Detailed characterization of the T box mechanism is essential to our long-term goal of developing novel antimicrobial agents that target this mechanism, which regulates essential genes in many Gram-positive pathogens. The T box system represents a unique molecular mechanism for RNA-mediated gene regulation, and a new function for tRNA in the cell. This study will provide information not only about this system, but also about RNA- mediated regulation and RNA-RNA recognition in a variety of cellular processes.